Immunomodulatory Effects of Histone Deacetylase 6 Inhibition in Suppressor Immune Cells in Multiple Myeloma

Abstract Abstract 128 The interaction of myeloma (MM) cells with bone marrow accessory cells and/or the extracellular matrix induces genomic, epigenomic and functional changes which promote tumor development, progression, cell adhesion mediated-drug resistance (CAM-DR), and immune suppression. To develop the most efficient anti-MM treatment strategy and prevent tumor escape from immune recognition, both enhancing anti-MM effector immune response and overcoming MM-induced immune suppression is essential. Suppressive immune cells including myeloid derived suppressor cells (MDSC), regulatory T cells (Treg) and IL-17 secreting Th (Th17) cells act as tumor promoters and suppressors of effector immune response, and therefore represent a significant barrier to current anti-tumor therapeutic strategies. Since, we and others have reported increased numbers of Treg and Th17 cells in MM, we here assessed MDSCs in both peripheral blood (PBMC) and bone marrow (BMMC) of patients with MM compared to healthy donors. Phenotypic analysis by flow cytometry showed a significant increase in CD14−CD11b+HLA-DRlowCD15+ MDSCs in both PBMC and BMMC from MM patients compared to healthy donors (p<0.01). Furthermore, coculture of MM cell lines with healthy PBMCs for 6 days demonstrated that MM cells significantly induce MDSC differentiation in healthy PBMCs (p<0.03). Recent studies have demonstrated that histone deacytlase 6 (HDAC6) is an important regulator of monocyte/macrophage-mediated immune response. We therefore next analysed the immunomodulatory effects of WT-161, a novel small molecule inhibitor of HDAC6, alone or in combination with lenalidomide (len) and bortezomib (bort), on suppressive immune cells in the MMBM microenvironment. To keep cell-cell interaction intact reflective of the MMBM microenvironment, PBMCs or BMMCs from MM patients were cultured in the absence or presence of WT-161 (0.5–5uM), len (1–10uM), and/or bort (2–5nM), and individual cell populations were analysed by flow cytometry. Phenotypic characterization of suppressive immune cells showed a significant decrease in both CD4+CD25+Foxp3+ Treg cells and MDSCs in MM-PBMCs and MM-BMMCs cultured with WT-161, alone or in combination with len or bort (p<0.01); however, there was no change in the expression of Th17 cells. To determine the functional mechanism of immune suppression, MDSC and Treg cells were isolated by magnetic-Ab sorting and cultured for 6 days with autologous T cells (TCR/IL-2 stimulated), with or without WT-161, len and bort, alone or in combination. T cell proliferation (by 3H-thymidine assay) was significantly inhibited in the presence of MDSCs, whereas WT-161 notably reversed MDSC-mediated T cell suppression. In contrast, len and bort did not show any significant effect. Intracellular reactive oxygen species (ROS, an MDSC-derived metabolic immune inhibitory molecule) expression was significantly decreased in MDSCs from MM cultured with WT-161, alone or together with len and bort (p<0.05). Additionally, WT-161 also reversed Treg-mediated T cell suppression as well as len. Cytokine profiling by intracellular flow cytometric analysis demonstrated that WT-161 significantly decreased IL-6 and GM-CSFR expression in MDSCs, whereas it induced IFNγ and IL-12 production in effector CD4T, CD8T and NKT cells. Finally, unstimulated or IL-2 prestimulated (36h) PBMCs or NK cells were cultured with MM cell lines (MM1.S, RPMI8226), in the absence or presence of WT-161 alone or with len and bort (4h), and anti-MM cytotoxic activity was determined by Cr51-release cytotoxicity assay. While len (48% killing) and WT-161 (39% killing) induced CTL-mediated cytotoxicity, WT-161 (53% killing) and len (56% killing) induced more potent NK cell-mediated anti-MM cytotoxicity. These data suggest that HDAC6 may have an immune regulatory function, and that inhibition of HDAC6 induces changes in suppressor immune cells leading to enhanced anti-MM immune response in MM microenvironment. Ongoing analysis of the effects of HDAC6 inhibition on immune cells in the tumor microenvironment will further define the role of HDAC6 in disease pathogenesis and suggest novel immune-based epigenetic-targeted therapies. Disclosures: Hideshima: Acetylon: Consultancy. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding. Bradner:Acetylon: Scientific Founder. Richardson:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Munshi:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy, Membership on an entity's Board of Directors or advisory committees. Anderson:Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees.

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Regulatory T Cell Response to Factor VIII in Mothers of Children with Hemophilia Inhibitors

Blood ◽

10.1182/blood.v126.23.3512.3512 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3512-3512

Author(s):

Margaret V. Ragni ◽

Lynn M. Malec ◽

Craig D. Seaman ◽

Lisa Butterfield

Keyword(s):

Immune Response ◽

T Cell ◽

Factor Viii ◽

Board Of Directors ◽

Research Funding ◽

Hemophilia A ◽

T Helper Cell ◽

T Helper ◽

Advisory Committees ◽

Healthy Donors

Abstract Background: Inhibitor formation is among the most serious complications of hemophilia A. These alloantibodies are directed against foreign infused factor VIII (FVIII) and occur in up to 25-30% of children with severe hemophilia A, after the first 9-10 exposures to FVIII. The inhibitor neutralizes infused FVIII, requiring alternative "bypass" therapy to treat bleeds, e.g. factor VIIa or FEIBA, to "bypass" the missing factor, and immune tolerance to suppress the inhibitor. Despite these approaches, bleeding is poorly controlled, resulting in 2-fold as many hospitalizations, 10-fold the cost, and 1.7-fold higher mortality. As the burden of disease is high, efforts have been directed at preventing inhibitors. There is increasing evidence that a specialized subset of T cells known as T-regs or CD4+/CD25+/FoxP3+ regulatory T cells, may play a role in mediating immune response to FVIII. Differentiation and function of T-regs are controlled by the X-chromosome-encoded transcription factor, FoxP3, and are generated centrally in the thymus and peripherally in extrathymic tissues. In the inhibitor-prone hemophilia A mouse (FVIII exon 16 knockout) T-regs reduce or prevent immune response to factor VIII: this is presumed to occur through T-reg-mediated hyporesponsiveness of T helper cells to factor VIII. T-regs also mediate maternal tolerance to the fetus in normal pregnancy. In early pregnancy there is an increase in immunosuppressive T-regs, peaking during the second trimester, and declining postpartum. T-regs are hypothesized to modify maternal immune response to the fetal "allograft" to promote tolerance to foreign paternal-derived antigens in the developing fetus. We hypothesized that defects in maternal T regulatory response to factor VIII might be associated with lack of normal FVIII tolerance in their offspring, leading to an alloantibody response, i.e. inhibitor formation. We therefore sought to quantitate and functionally characterize T-regs in mothers of children with congenital hemophilia A with inhibitors, in order to determine if maternal lack of tolerance to FVIII is associated with inhibitor formation in their affected sons. Methods: Following informed consent, heparinized tubes were drawn from four mothers of hemophilic children, two with and two without inhibitors, cared for at the Hemophilia Center of Western PA (HCWP). T-reg responses to factor VIII were quantitated by flow cytometry, and phenotyping was performed by staining with anti-CD4/Foxp3/CD3/CD25/CD39 monoclonal antibody markers. Functional characteristics of the T-regs were determined in a T-reg proliferation assay using autologous antigen presenting cells (DCs) in the presence or absence of FVIII. Results: CD4+ T cell proliferative responses to factor VIII (FVIII) were decreased in all four mothers (Figure 1). Mothers of hemophilic sons with inhibitors demonstrated a 1.36-fold decrease (Pt 001) and a 1.1-fold decrease (Pt 002), as compared with mothers of hemophilic sons without inhibitors, who demonstrated a 1.47-fold decrease (Pt 003) and a 1.45-fold decrease (Pt 005) in CD4+ proliferative responses, unlike the two healthy donors who showed minimal (or no) factor VIII response. Patients showed slightly less proliferation with Factor VIII vs. no antigen; healthy donors showed minimal (or no) Factor VIII response (Figure 1). Discussion: These preliminary results demonstrate lower T cell proliferative responses to FVIII in mothers of children with inhibitor patients than in mother of children without inhibitors. This suggests there is lack of tolerance to FVIII in mothers of children with hemophilia, with or without inhibitors. These responses would also imply these mothers have no tolerance to paternal FVIII, but further studies will be needed to confirm these findings and to characterize maternal T regulatory response to factor VIII, and, in particular, paternal FVIII. Figure 1. Percent CD4+ T Helper Cell Proliferation +/- Factor VIII (FVIII) Figure 1. Percent CD4+ T Helper Cell Proliferation +/- Factor VIII (FVIII) Disclosures Ragni: Alnylam: Research Funding; Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding; Biogen: Research Funding; SPARK: Research Funding; Pfizer: Research Funding; Ferring Pharmceuticals: Research Funding; National Hemophilia Foundation: Membership on an entity's Board of Directors or advisory committees; Medscape, Web MD: Honoraria; Genentech Roche: Research Funding; Vascular Medicine Institute: Research Funding; Foundation Women Girls Blood Disorders: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tacere Benitec: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding; CSL Behring: Research Funding; Dimension Therapeutics: Research Funding; Biomarin: Research Funding. Malec:Biogen: Research Funding; Baxalta: Research Funding. Seaman:Vascular Medicine Institute: Research Funding.

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T Cell Immunity Toward Viral- and Tumor-Antigens Is Preserved in MDS Patients

Blood ◽

10.1182/blood-2019-126792 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4225-4225

Author(s):

Hussein Hamad ◽

Wingchi K Leung ◽

Spyridoula Vasileiou ◽

Shivani Mukhi ◽

Quillan Huang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Healthy Donor ◽

Tumor Antigens ◽

Ex Vivo ◽

Advisory Committees ◽

Cell Immunity ◽

Healthy Donors ◽

T Cell Lines

Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders characterized by bone marrow failure and a propensity to progress to acute myeloid leukemia (AML). A core component of the underlying pathogenesis in MDS is deregulation of inflammatory cytokines, such as tumor growth factor-β (TGFβ), which impact the function of immune cells and hence their capacity to mount anti-infective or anti-tumor responses. However, little is known about antigen-specific T cell function in patients with MDS. We hypothesized that virus-specific T cell (VST) function might be preserved in patients with MDS, and that the functional capacity of T cells reactive against tumor-associated antigens aberrantly overexpressed by clonal MDS cells such as Cyclin A1 (CCNA1) and Proteinase (PR3) might also be preserved and exploited for immunotherapeutic purposes. Following informed consent, we collected peripheral blood samples from 10 patients (pts) with MDS and 17 healthy donors. Most pts (9 out of 10) were transfusion dependent and 3 subsequently underwent an allogeneic HSCT. Table 1 summarizes the other clinical characteristics, karyotypic and mutational profile at the time of blood collection. Compared with T cells isolated from healthy donors, MDS patient-derived T cells had a similar CD4 to CD8 ratio (1.5-2.5:1 for healthy donors and 3:1 for MDS pts), but displayed a more exhausted profile at baseline (CD3+TIM3+: 1% in healthy donors and 5% in MDS pts) and produced higher levels of inflammatory cytokines [IFNγ (18±3pg/ml vs 36±16pg/ml, healthy donor vs MDS; p=0.12), and IL-8 (56±32 vs 704±446 pg/ml, p=0.01)]. Next, to assess the capacity of MDS pts to mount ex vivo functional virus-directed responses, we stimulated patient-derived PBMCs (n=5) with overlapping peptide libraries (pepmixes) spanning immunogenic AdV, CMV, EBV, BK and HHV-6 antigens. Similar to healthy donor-derived T cell lines (n=5, 3 specific for 4 viruses and 2 for 5 viruses), all 5 MDS patient-derived lines demonstrated specificity for one or more of the target viruses (1 for 5 viruses, 1 for 4, 2 for 3 and 1 for 1 virus) as observed by IFNγ ELISpot assay with comparable magnitude (range Adv: 43-730 vs 384-941 in healthy donors, CMV: 0-1599 vs 0-3002, EBV: 0-1486 vs 0-541, BK: 0-839 vs 38-275 and HHV6: 0-794 vs 5-407 SFU/2x105 cells, respectively). We next examined the feasibility of expanding autologous MDS-antigen directed T cell products (n=10) to determine whether an adoptive immunotherapeutic approach might be applicable for MDS treatment. Thus, we exposed patient-derived PBMCs to autologous dendritic cells (DC) loaded with pepmixes spanning 6 MDS-associated antigens (CCNA1, survivin, WT1, PRAME, PR3 and NYESO1). After 3 rounds of stimulation, the products obtained were predominantly CD3+ T cells (mean 88±1.3%) that were polyclonal (CD4: 46±5% and CD8: 41±4%) containing predominantly memory T cells (TEM: 36±6% TCM: 37±5% and Tnaïve =13±3%). Six lines (60%) showed specific recognition to at least one of the target antigens: 4 lines specific for PRAME, 1 for CCNA1, 1 for WT1 and 1 for NYESO1 (range 0-40, 0-184, 0-1386 and 0-179 SFU/2x105 cells, respectively by IFNγ ELIspot). T cell lines were capable of specifically secreting multiple effector cytokines in response to targets (TNFα: 12% and IFNγ: 16% in response to PRAME in a representative patient-derived T cell line). Furthermore, this line was capable of killing PRAME+ targets in a 4hr 51Cr release assay [60% specific lysis, E:T 20:1]. In conclusion, functional virus-directed T cell immunity in patients with MDS is preserved, potentially explaining the lower rates of viral reactivation seen in these patients compared with other infections. Moreover, T cells specific for MDS-expressed tumor antigens can also be successfully expanded ex vivo from patients. Taken together this raises the possibility of applying an adoptive immunotherapeutic approach for the treatment of MDS. Disclosures Ramos: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding. Leen:Allovir: Consultancy, Other: Cofounder, Ownership Interest; Marker Therapeutics: Consultancy, Other: Cofounder, Ownership Interest.

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Cellular Immune Responses To Vector In a Gene Therapy Trial For Hemophilia B Using An AAV8 Self-Complementary Factor IX Vector

Blood ◽

10.1182/blood.v122.21.717.717 ◽

2013 ◽

Vol 122 (21) ◽

pp. 717-717

Author(s):

Etiena Basner-Tschakarjan ◽

Federico Mingozzi ◽

Yifeng Chen ◽

Amit Nathwani ◽

Edward Tuddenham ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Factor Ix ◽

High Dose ◽

Advisory Committees ◽

Ifn Γ ◽

Cellular Immune

Abstract In a clinical study of gene transfer for hemophilia B an adeno-associated virus vector serotype 8 (AAV8) expressing a self-complementary liver-specific expression cassette for the factor IX (FIX) transgene was administered intravenously in ten affected subjects. The results of the first part of the study have been published (NEJM 365:2357-65, 2011). In this abstract we present the immunomonitoring data, using Interferon-gamma (IFN-γ) ELISpot and polyfunctional T cell analysis of peripheral blood mononuclear cells (PBMCs) to monitor cellular immune responses to vector capsid and to Factor IX. We have previously shown that the cellular immune response was directed solely towards AAV capsid epitopes, not FIX, and that the response was dose-dependent. Out of six subjects infused in the high dose cohort (2x1012vg/kg), 4/6 manifested a minor rise in liver enzyme levels and detection of capsid-specific T cell reactivitiy in the ELISpot assay at ∼7-10 weeks post vector infusion. Maximum results on IFN- γ ELISpots ranged from 200-500 sfu/million cells. In two of these cases a modest decline in FIX level also occurred. Prompt initiation of prednisolone reversed these effects and rescued FIX levels. The remaining two subjects infused at the high dose, showed no rise in liver enzyme levels at any time point. However capsid reactive T cells were detectable in one subject as early as one to two weeks after vector infusion in peripheral blood by IFN-γ ELISpot assay, while no activation at all was detected in the other subject, possibly due to low cell recovery and viability of the cells. A similar immune response profile, with early detection of activated T cells but no rise in liver enzymes, was also observed in both subjects in the intermediate dose cohort in the first part of this study. Polyfunctional T cell analysis revealed concurrent Interleukin-2, Tumor necrosis factor-alpha and CD107a positivity in activated T cells at the peak of activation. Furthermore it showed that capsid-specific early T cell responses were detectable in the CD4+ T cell and later in the CD8+T cell compartment. Long-term immune monitoring of all subjects is ongoing. Importantly in one of the first two subjects treated at the high dose, capsid reactive T cells were detected by ELISpot 1.5 years after gene transfer; these cells were not detected in the other subject in whom long-term follow-up samples are available. Of note, capsid-reactive T cells were also seen at late time points (>1 year after infusion) in a middle dose subject and a low dose subject. Despite detectable T cell reactivity towards the AAV capsid in the peripheral blood FIX expression remained stable, suggesting that there is a short window of time during which transduced hepatocytes present a target for cytotoxic T cells, and that T cell positivity after this window is without any clinical consequences. In conclusion, for this scAAV8 vector there appears to be a critical threshold vector dose for a clinically detectable immune response, starting at 2x1012 vg/kg. The clinically detectable response occurred in four out of six subjects so far, and was manifest within a critical time interval of 7-10 weeks post infusion. The capsid-specific response was polyfunctional and detected in CD4+ and CD8+T cells in peripheral blood. It is important to note that not all subjects treated at the high dose developed an immune response. However, given the limited dataset, it is not yet possible to define predictive parameters, e.g. HLA type of a subject, for an immune response. Continued monitoring and future studies with more subjects will be necessary to confirm the presented findings, in particular time and rate of occurrence of a cellular response as well as successful treatment with a short course of Prednisolon. Disclosures: Tuddenham: Pfizer: Consultancy. Reiss:Hemophilia of Georgia: Honoraria. High:BristolMyersSquibb: Consultancy, membership on a Data Safety and Monitoring Board, membership on a Data Safety and Monitoring Board Other; Elsevier, Inc.: royalties from textbook, royalties from textbook Patents & Royalties; Genzyme, Inc.: Membership on an entity’s Board of Directors or advisory committees; Intrexon: Consultancy; Novo Nordisk: Consultancy, Member of a grant review committee, Member of a grant review committee Other; Shire : Consultancy; Benitec: Consultancy; bluebirdbio, Inc.: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; BioMarin: Consultancy; Alnylam Pharmaceuticals: Consultancy, Membership on an entity’s Board of Directors or advisory committees.

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Early Changes in Circulating T-Cell Immune Profiles in Patients with Relapsed Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma: Data from the Phase 3, Double-Blind HELIOS Trial

Blood ◽

10.1182/blood.v128.22.4397.4397 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4397-4397 ◽

Cited By ~ 1

Author(s):

Rajendra Damle ◽

Michael Schaffer ◽

Shalini Chaturvedi ◽

Charles Phelps ◽

Regina Aquino ◽

...

Keyword(s):

T Cell ◽

Board Of Directors ◽

T Cell Activation ◽

Cell Activation ◽

Treg Cells ◽

Lymphocytic Leukemia ◽

T Cell Subsets ◽

Phase 3 ◽

Advisory Committees ◽

Double Blind

Abstract Introduction Ibrutinib is a first-in-class, once-daily, oral, covalent inhibitor of Bruton's tyrosine kinase (BTK) that as a single agent has significantly improved overall survival in patients (pts) with both treatment naïve and relapsed/refractory chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). In the phase 3 HELIOS trial, the addition of ibrutinib to bendamustine + rituximab (BR) resulted in an 80% reduction in disease progression or death (HR, 0.203, 95% CI, 0.150-0.276; p < 0.0001) and confirmed for the first time, in a randomized setting, the benefit of ibrutinib-based therapy compared with standard chemoimmunotherapy in previously treated pts (Chanan-Khan, et al. Lancet Oncol. 2016;17(2):200-11). Independent Review Committee assessed progression-free survival (PFS) at 18 months was 79% (95% CI: 73%-83%) in the ibrutinib +BR (I+BR) arm and 24% (95% CI: 18%-31%) in the placebo + BR (P+BR) arm. While the activity of ibrutinib is primarily in B-cells where it inhibits BTK-dependent survival pathways, it may also have significant effects on the immune system by exerting an effect on T-cells (by inhibition of interleukin-2-inducible kinase and related shift from Th2 to Th1-based immunity, which in turn affects Treg and Th17 activity) and via normalization of immune function, thus affecting tumor response. Here, we report changes in circulating T-cell immunophenotypes in a subset of pts treated with either I+BR or P+BR in the HELIOS study. Methods HELIOS is a randomized, double-blind, placebo-controlled, phase 3 study. Pts with active CLL/SLL following ≥ 1 prior therapy were randomized 1:1 to receive BR (≤ 6 cycles) with either ibrutinib (420 mg daily; n = 289) or placebo (n = 289). Pts with del17p (> 20% of cells) were excluded. Peripheral blood was collected at the start of the study, at Cycle 1, Day 15 (C1D15) and at the end of treatment/time of progressive disease (EOT/PD). From these samples, peripheral blood mononuclear cells (PBMC) were separated and cryopreserved. PBMC were subjected to flow-cytometric analysis of T-cell subsets including CD4, CD8 and markers indicative of Treg cells (CD25+CD127low) and Th17 cells (CCR4+CCR6+). In addition, expression of T-cell checkpoints ICOS, PD-1 and OX-40 by CD4 and CD8 cells were evaluated as an indicator of cell activation or activation/exhaustion. The findings from the HELIOS study reported here are based on analysis of paired samples (C1D1 and C1D15) of 29 CLL cases from the I+BR arm and 22 cases from the P+BR arm. The Mann-Whitney U test was applied to determine the significance of the differences in each of T-cell subtype between the two arms. Results Overall, there was a net increase in the percentage of CD3+ cells with treatment in both the I+BR-treated pts and P+BR-treated pts, which was observed as early as Day 15 (+12.0% and +11.42% of total lymphocytes, respectively, at C1D15 compared with C1D1; Figure 1). The EOT/PD samples showed a continued increase in the percentage of CD3+ cells in the I+BR pts, the majority of whom (11/12) were responders, while the opposite trend was observed in the P+BR patients (8/21 responders). Focusing on the early changes in the analysis of T-cell subsets, a pronounced decrease in the percentage of Th17 CD4+ cells was noted in the I+BR pts (mean: -2.49%) but in the P+BR pts, an increase of this subset (mean: +2.66%) was observed (p = 0.011; Figure 2). When comparing other CD4+ subsets, decreases in Treg cells were seen in both pt groups, with a corresponding increase in the T-cell activation marker ICOS. Less significant changes were observed in the other T-cell markers studied, however, a trend to increased PD-1 was seen in the P+BR pts but not in the I+BR pts. The safety profile in these pts was consistent with previous reports for the study. Conclusions These results suggest that the addition of ibrutinib to BR helped restore T-cell proportions in this subset of cases and it is noteworthy that this effect was visible even within 15 days of initiation of therapy. Data from the I+BR pts showed a pronounced decrease in the percentage of Th17 CD4+ cells, which are primarily pro-tumorigenic but may sometimes have anti-tumor effects, and a concomitant decrease in the percentage of Treg cells, which may explain the overall increase in T-cell activation. These findings may also be related to the observed increase in PFS with the addition of ibrutinib to BR reported in the trial. Disclosures Damle: Janssen Research & Development: Employment. Schaffer:Janssen Research & Development: Employment. Chaturvedi:Janssen Research & Development: Employment. Phelps:Johnson & Johnson: Employment, Equity Ownership. Aquino:Janssen Research & Development: Employment. Mahler:Janssen Research & Development: Employment. Salman:Janssen Research & Development: Employment, Equity Ownership, Other: Travel, Accommodations, Expenses. Howes:Janssen Research & Development: Employment. Loscertales:Janssen Research & Development: Consultancy; Roche: Consultancy; Gilead: Consultancy; AbbVie: Consultancy. Trneny:Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Research & Development: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Balasubramanian:Janssen Research & Development: Employment.

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Gabarap Loss Mediates Immune Escape in High Risk Multiple Myeloma

Blood ◽

10.1182/blood-2021-148621 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 891-891

Author(s):

Annamaria Gulla ◽

Eugenio Morelli ◽

Mehmet K. Samur ◽

Cirino Botta ◽

Megan Johnstone ◽

...

Keyword(s):

Immune Response ◽

T Cell ◽

Clinical Outcome ◽

Board Of Directors ◽

Research Funding ◽

Immune Escape ◽

Publicly Traded ◽

Advisory Committees

Abstract Immune therapies including CAR T cells and bispecific T cell engagers are demonstrating remarkable efficacy in relapsed refractory myeloma (MM). In this context, we have recently shown that proteasome inhibitor bortezomib (BTZ) results in immunogenic cell death (ICD) and in a viral mimicry state in MM cells, allowing for immune recognition of tumor cells. Induction of a robust anti-MM immune response after BTZ was confirmed both in vitro and in vivo: treatment of 5TGM1 MM cells with BTZ induced tumor regression associated with memory immune response, confirmed by ELISPOT of mouse splenocytes. We have confirmed the obligate role of calreticulin (CALR) exposure in phagocytosis and the ICD process, since BTZ-induced ICD is impaired in CALR KO MM cells both in vitro and in vivo. We further showed that the therapeutic efficacy of BTZ in patients was correlated with ICD induction: BTZ-induced ICD signature was positively correlated with OS (p=0.01) in patients enrolled in the IFM/DFCI 2009 study. Together, these studies indicate that ICD is associated with long-term response after BTZ treatment. In this work, we reasoned that genomic or transcriptomic alterations associated with shorter survival of MM patients after BTZ treatment may impair activation of the ICD pathway. To this aim, we performed a transcriptomic analysis of purified CD138+ cells from 360 newly diagnosed, clinically-annotated MM patients enrolled in the IFM/DFCI 2009 study. By focusing on genes involved in the ICD process, we found that low levels of GABA Type A Receptor-Associated Protein (GABARAP) were associated with inferior clinical outcome (EFS, p=0.0055). GABARAP gene locus is located on chr17p13.1, a region deleted in high risk (HR) MM with unfavorable prognosis. Remarkably, we found that correlation of low GABARAP levels with shorter EFS was significant (p=0.018) even after excluding MM patients with del17p; and GABARAP is therefore an independent predictor of clinical outcome. GABARAP is a regulator of autophagy and vesicular trafficking, and a putative CALR binding partner. Interestingly, among a panel of MM cell lines (n=6), BTZ treatment failed to induce exposure of CALR and MM cell phagocytosis by DCs in KMS11 cells, which carry a monoallelic deletion of GABARAP. This effect was rescued by stable overexpression of GABARAP. Moreover, CRISPR/Cas9-mediated KO of GABARAP in 3 ICD-sensitive cell lines (AMO1, H929, 5TGM1) abrogated CALR exposure and ICD induction by BTZ. GABARAP add-back by stable overexpression in KO clones restored both CALR exposure and induction of ICD, confirming GABARAP on-target activity. Similarly, pre-treatment of GABARAP KO cells with recombinant CALR restored MM phagocytosis, further confirming that GABARAP impairs ICD via inhibition of CALR exposure. Based on these findings, we hypothesized that GABARAP loss may alter the ICD pathway via CALR trapping, resulting in the ICD resistant phenotype observed in GABARAP null and del17p cells. To this end, we explored the impact of GABARAP KO on the CALR protein interactome, in the presence or absence of BTZ. Importantly, GABARAP KO produced a significant increase of CALR binding to stanniocalcin 1 (STC1), a phagocytosis checkpoint that mediates the mitochondrial trapping of CALR, thereby minimizing its exposure upon ICD. Consistently, GABARAP KO also affected CALR interactome in BTZ-treated cells, which was significantly enriched in mitochondrial proteins. Importantly, co-IP experiments confirmed GABARAP interaction with STC1. These data indicate a molecular scenario whereby GABARAP interacts with STC1 to avoid STC1-mediated trapping of CALR, allowing for the induction of ICD after treatment with ICD inducers; on the other hand, this mechanism is compromised in GABARAP null or del17p cells, and the STC1-CALR complex remains trapped in the mitochondria, resulting in ICD resistance. To functionally validate our findings in the context of the immune microenvironment, we performed mass Cytometry after T cell co-culture with DCs primed by both WT and GABARAP KO AMO1 clones. And we confirmed that treatment of GABARAP KO clones with BTZ failed to activate an efficient T cell response. In conclusion, our work identifies a unique mechanism of immune escape which may contribute to the poor clinical outcome observed in del17p HR MM patients. It further suggests that novel therapies to restore GABARAP may allow for the induction of ICD and improved patient outcome in MM. Disclosures Bianchi: Jacob D. Fuchsberg Law Firm: Consultancy; MJH: Honoraria; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Richardson: AstraZeneca: Consultancy; Regeneron: Consultancy; Protocol Intelligence: Consultancy; Secura Bio: Consultancy; GlaxoSmithKline: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; Takeda: Consultancy, Research Funding; AbbVie: Consultancy; Karyopharm: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding; Oncopeptides: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding. Chauhan: C4 Therapeutics: Current equity holder in publicly-traded company; Stemline Therapeutics, Inc: Consultancy. Munshi: Legend: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Bristol-Myers Squibb: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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Proteasome Inhibitors Impair the Innate Antiviral Immune Response and Potentiate Pelareorep-Based Viral Therapy in Multiple Myeloma

Blood ◽

10.1182/blood-2019-128740 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1816-1816

Author(s):

Ada Dona' ◽

Domenico Viola ◽

Enrico Caserta ◽

Francesca Besi ◽

James F Sanchez ◽

...

Keyword(s):

Immune Response ◽

T Cell ◽

Board Of Directors ◽

Ex Vivo ◽

Phase 1 ◽

Single Agent ◽

Advisory Committees ◽

Antiviral Immune Response ◽

Myeloma Cells ◽

First Time

INTRODUCTION: Pelareorep is the infusible form of human reovirus (RV). Our single-agent phase 1 RV trial in relapsed multiple myeloma (MM) showed that pelareorep treatment selectively infected MM cells, as viral RNA was found in myeloma cells and not the bone marrow (BM) stroma. However, we did not observe apoptosis. Our ongoing phase 1 trial, which combines the proteasome inhibitor carfilzomib with RV, has demonstrated RV infection, apoptosis, and clinical responses. We investigated the molecular mechanisms behind the role of a PI (carfilzomib) in this setting. RESULTS: In all MM cell lines we tested (n=4), independently from their sensitivity to RV infection (Stiff et al., Mol Cancer Ther, 2016), viral replication and apoptosis was impaired when MM cells were directly exposed to PIs (carfilzomib and bortezomib). When this experiment was repeated in the setting of the bone marrow cellular fraction or peripheral blood mononuclear cells (PBMCs), it had the opposite effect, as the addition of PI to RV increased RV replication and apoptosis in MM cells. When we washed PBMCs after overnight exposure to RV+PI or to either single agent, then added MM cells, we observed higher infection and apoptosis in cancer cells co-cultured with RV+PI compared to levels from PBMCs treated with each of the single agents, suggesting that PIs increase the ability of PBMCs to serve as a reservoir for infectious reovirus. Monocytes (CD14+) can engage in phagocytosis of reovirus (Berkeley et al., Cancer Immunol Res, 2018), and accordingly we found that RV genome and capsid protein production were detected in CD14+ cells, but not in CD14-depleted PBMCs, and increased upon PI treatment compared to that in RV-treated CD14+ cells. Given that the NF-κB complex is a key proinflammatory signaling pathway associated with the early innate-antiviral immune response, and because PIs can block the degradation of the NF-κB inhibitor IκBα upon phosphorylation, we investigated the effect of the specific IκBα inhibitor Bay-11 in RV viral replication. Our data show that either PI or Bay-11 can inhibit RV-induced IκBα phosphorylation and its subsequent degradation upon RV infection in CD14+ cells, an effect associated with higher capsid formation in RV-treated CD14+ cells in combination with PI or Bay-11, compared to levels from RV alone. Cytokine profiling in PBMCs and CD14+ cells treated with RV in combination with either PI or Bay-11 showed a significant decrease in IFN-α and IFN-β (IFNs) levels and a concomitant increase in RV replication, in contrast to levels from RV alone (p<0.001). We then decided to investigate whether CD14 depletion could affect RV delivery to the cancer cells invivo. Upon intra-femoral injection of 5TGM1 MM cells into syngeneic C57BL/KaLwRij mice, the mice in which the monocytes were depleted by clodronate-liposome treatment before intravenous RV injection showed lower capsid protein formation in the BM MM cells compared to that in mice where the monocytic population was intact. Because monocytes respond to infection by dividing into macrophages to eliminate pathogens, we wanted to investigate whether PI could impair this effect. Intriguingly, although higher levels of viral genome were detected in PI+RV-treated CD14+ cells compared to RV-treated cells (p<0.01), PI abolished ex-vivo RV-induced monocyte differentiation (n=3, p<0.001), without affecting the ability of RV-infected CD14+ cells to induce higher MM cell killing as compared to that from the CD14+ fraction treated with either single agent. Immune killing assays showed that RV-infected MM cells are more susceptible than non-infected cells to T cell effectors (p<0.001), a mechanism that is further potentiated by the addition of PI. In fact, in contrast to the PI-induced blocking of monocyte expansion and activation in response to the virus (n=3, p<0.001), our data suggest that the PI potentiate the expansion of CD8+ T cells, both ex vivo and in two RV+PI treated patients we analyzed so far. CONCLUSIONS: Here we report for the first time that PIs enhance pelareorep entry, infection, and killing of myeloma cells through its effect on the CD14+ fraction. Reovirus infection and replication within CD14+ cells are augmented by PI-induced NF-κB inhibition of the early innate pro-inflammatory immune response. We also report for the first time that carfilzomib induces direct T cell activation and potentiates T cell killing activity against RV-infected MM cells. Disclosures Krishnan: Takeda: Research Funding; Celgene, Z Predicta: Other: Stock Ownership; Amgen, Takeda: Speakers Bureau; Sutro BioPharma, zPredicta: Consultancy; Celgene, Janssen, Sanofi, BMS: Consultancy. Sborov:Celgene: Honoraria; Janssen: Consultancy. Hofmeister:Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Imbrium: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees.

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Poor Clinical Outcome in Pediatric Immunotherapy Is Mediated By a Pre-Existing Overactive IL-18-IFNy Immune Phenotype

Blood ◽

10.1182/blood-2021-148013 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 168-168

Author(s):

Katelyn Burleigh ◽

Anna Kus ◽

Alice Long ◽

Michael C. Jensen ◽

Seth Masters ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Early Time ◽

Advisory Committees ◽

Time Points ◽

The Poor ◽

Healthy Donors ◽

Car T ◽

Group 2

Abstract Background: CAR-T cells have revolutionized the treatment of relapsed B-cell acute lymphoblastic leukemia (ALL), however the field is plagued by 50% relapse, 10-15% non-response, 20% severe toxicity (cytokine release syndrome (CRS) and neurotoxicity) rates. Studies on CAR patient outcomes thus far have largely focused on the contribution of characteristics of the CAR product and post-CAR symptoms 1-8, largely overlooking the potential role of the patient's pre-CAR immunophenotype beyond T-cells in driving therapeutic response and toxicity. We hypothesized that a patient's inflammatory status prior to immunotherapy could be used clinically to inform interventional and therapeutic mechanistic strategies. Taking inspiration from the clinical presentation overlap between CRS and hemophagocytic lymphohistocytosis (HLH), we hypothesized a common pathophysiology. IL-18 signaling in HLH disorders is well known to play a causative role in anomalous T-cell function 9. Importantly, IL-18 is also linked to tumor regression and progression 10-13. This coupled with the poor overall 5-year survival of secondary HLH patients associated with malignancies 14,15 suggested to us that an overactive IFNγ (T-cell)-IL-18 (myeloid) axis may also drive post-CAR toxicity, as well as overall therapeutic efficacy. Methods: To test whether patient-specific immunity and an overactive IFNy-IL-18 axis could predict toxicity, response, and survival, we applied a machine learning classifier to cytokine and cell-type profiles of blood samples obtained to prior and at early time points following CD19 CAR T cell infusion of retrospective cohort based on response/non-response, across the continuum of toxicity severity (both CRS and neurotoxicity) and matched availability of samples from 30 of 43 patients on the PLAT phase1 trial (NCT02028455) 2,16. Results/Discussion: We found evidence for a pre-existing overactive HLH-like axis in some patients; this included both patients with no response to CAR-T therapy as well as patients who experienced high levels of toxicity and diminished long-term survival. Stratification based on this axis was significantly associated with survival rates - Group 2 patients with high levels of IFNy-IL-18 had a median survival of 16.8 months post-CAR, (versus >48 months post-CAR, Group 1). Importantly, we identified an immune sub-type (CD161+IFNy+ T cells which are hyper responsive to IL-18) that is increased in this poor survival Group 2 prior to therapy, which suggests that patients could be effectively stratified prior to CAR infusion. Given the poor clinical outcome associated with high levels of CD161+IFNy+ T cells, we hypothesized that high levels may be a marker for primed pro-inflammatory myeloid activation, independent of cancer. Thus, we reasoned that donor monocytes from healthy donors with high levels of CD161+IFNy+ T cells could be induced with continued interferon signaling/priming into a Group 2 low survival/neurotoxicity phenotype. Healthy donors with elevated CD161+IFNy+ T cells had increased pro-inflammatory potential including IL-18 signaling in response to IFNy prime. We drove healthy donors to an elevated CD161+ IFNy phenotype utilizing an IL-12 prime, suggesting that patents with high levels of CD161+ cells had a monocytic priming event (e.g., pathogenic exposure, genetic predisposition, vaccination, etc.) prior to therapy that drove the poor clinical outcomes. Based on this data, we propose that a child's cytokine pre-inflammatory status might be independent of cancer and drives immunotherapeutic response and therefore tumor response and toxicity. Conclusions: In summary, we have identified that an HLH-like signaling axis pre- and early time points post- CAR therapy stratify patient outcomes predicting the onset of poor clinical outcomes prior to their clinical onset such as neurotoxicity, non-response, and relapse. We have also identified biomarker signatures prior and at early time points post-CAR such as cytokines (IFNγ-IL-18 signaling) and immune cell subsets (CD161+ IFNγ+ cells) responsible for the HLH-like signaling axis that could serve as potential future interventional targets. Finally, this study highlights the need to study patient-specific immune characteristics prior to CAR therapy, as these may be predictive of CAR success and failure and may help to identify novel therapeutic approaches and targets improving clinical outcomes. Figure 1 Figure 1. Disclosures Burleigh: ShapeTx: Current holder of stock options in a privately-held company. Jensen: BMS: Patents & Royalties; Umoja Biopharma: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bluebird Bio: Research Funding. Masters: IFM therapeutics: Membership on an entity's Board of Directors or advisory committees. Vince: Avammune Therepeutics: Membership on an entity's Board of Directors or advisory committees; Exopharm: Membership on an entity's Board of Directors or advisory committees. Gardner: Novartis: Consultancy; BMS: Patents & Royalties.

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Immunomodulatory EFFECTS of Lenalidomide and Pomalidomide ON INTERACTION of TUMOR and BONE MARROW Accessory CELLS IN MULTIPLE MYELOMA.

Blood ◽

10.1182/blood.v114.22.950.950 ◽

2009 ◽

Vol 114 (22) ◽

pp. 950-950

Author(s):

Gullu Gorgun ◽

Elisabetta Calabrese ◽

Teru Hideshima ◽

Giulia Perrone ◽

Giada Bianchi ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Immune Cells ◽

Research Funding ◽

Effector Cell ◽

Cytokine Signaling ◽

Advisory Committees ◽

Effector Cells ◽

Healthy Donors ◽

Socs3 Expression

Abstract Abstract 950 The bone marrow (BM) microenvironment consists of extracellular matrix and the cellular compartment including bone marrow stromal cells (BMSCs) and immune cells. Interaction between multiple myeloma (MM) cells and BM cells induces growth, survival, migration, and drug resistance in MM, via both cell-cell contact and cytokines. Even though MM cell interaction with BMSCs has been extensively studied, the role of immune cells in the MM BM milieu is not yet defined. The IMiDs® immunomodulatory agents lenalidomide (len) and pomalidomide (pom) target not only MM cells, but also MM cell-immune cell interactions and cytokine signaling. For example, we and others have shown that len stimulates T cell proliferation, secretion of IL2 and IFNγ, as well as promotes CTL and NK cell activity against MM cells. Here we examined the in vitro immunomodulatory effects of len or pom on cytokine signaling triggered by interaction of effector immune cells with MM cells and BMSCs. PBMCs or BMMNCs obtained from patients with rel/ref MM or healthy donors after informed consent. PBMCs were cultured either alone or with BMSC, in the absence or presence of len (1μM) or pom (1μM) for 1-48h. To determine whether len or pom regulate cytokine signaling in effector cells, we used flow cytometry to analyze their effects on suppressor of cytokine signaling proteins (SOCS, including SOCS1, SOCS2, SOCS3, CIS) expression in effector cells from both healthy donors and patients with MM. Len or pom diminished IL2 and IFNγ regulators SOCS1 and SOCS3 expression in effector cells from both BM and PB of MM patients. Additionally, coculture of MM cell lines, MM1S, U266, OPM1, RPMI, LR5 and DOX40, with healthy PBMCs induced SOCS1 and SOCS3 expression in effector cells; conversely, treatment with len or pom downregulated the SOCS1 and SOCS3 expression in effector cells. To assess effects of immunomodulatory agents on immune cell proliferation in their milieu, healthy or MM-PBMCs and MM-BMMNCs were prelabeled with CFSE and stimulated with PHA (5μg/ml) or anti-CD3 (1μg/ml) in the absence or presence of len or pom for 7 days. The proliferation of CD4T and CD8T, NKT, and NK cells was assessed by CFSE flow cytometric analysis. Len or pom induced CD4T cell (%Divided: Cont:55, len or pom >72), CD8 T cell (%Div: Cont:34, len or pom>60) and NKT cell (%Div: Cont:3.5, len or pom >8) proliferation, as well as stimulated IL2 (2-4 fold) and IFNγ (2 fold) production in effector cells from MM. It has been demonstrated that SOCS1 gene negatively regulates IL6 signaling and is silenced by methylation in MM cells. To understand the mechanism of cytokine inhibitory signaling in both effector cells and MM cells, we next analysed the interaction of effector cell with MM cells that were epigenetically modified to express SOCS1. SOCS1 methylation in MM cells was confirmed by SOCS1 gene methylation-specific polymerase chain reaction (SOCS1-MSP). Genomic DNA was isolated from MM cell lines (MM1S, RPMI8226, OPM1, INA6 and U266), sodium bisulfite-modified, and then subjected to MSP using MSP primers that specifically recognize unmethylated or methylated SOCS1 gene. SOCS1 gene was methylated and resulted in silenced SOCS1 protein expression in all MM cell lines. To delineate the role of SOCS in effector cell response against MM cells, MM cell specific cytotoxic T lymphocytes (CTL) were generated. T cells from healthy donors were stimulated with dendritic cells pulsed with apoptotic bodies of MM1S or U266 cells for 4 weeks, and cytotoxicity was measured by standard 51Cr-release assay. To reverse SOCS1 methylation, target MM cells were cultured with 5'-Azacytidine (Aza) or trichostatin A (TsA), alone or in combination with len or pom. CTLs were pretreated with len or pom for 24h and cocultured with DNA-modified or unmodified 51Cr-labeled target cells. Len induced more potent CTL response against MM cells that were treated with len and Aza combination (83% specific killing) than len alone (%50 specific killing). Len also showed more potent anti-MM activity, assessed by 3[H]thymidine proliferation assay, in the presence of Aza than alone (p<0.05). These data demonstrate that modulation of SOCS genes by blocking BMSC derived inhibitory cytokine signaling may enhance effector cell response and promote efficacy of len or pom in MM. Ongoing analysis of effects of len or pom on immune cells in the BM environment will both define their role in disease pathogenesis and suggest novel immune-based targeted therapies. Disclosures: Munshi: Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium: Research Funding; Novartis: Research Funding; Celgene: Research Funding.

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Myeloid Derived Suppressor Cells (MDSCs) Regulate Tumor Growth, Immune Response and Regulatory T Cell (Treg) Development in the Multiple Myeloma Bone Marrow Microenvironment

Blood ◽

10.1182/blood.v120.21.565.565 ◽

2012 ◽

Vol 120 (21) ◽

pp. 565-565

Author(s):

Gullu Topal Gorgun ◽

Gregory Whitehill ◽

Jennifer Lindsey Anderson ◽

Teru Hideshima ◽

Jacob P. Laubach ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Tumor Development ◽

Suppressor Cells ◽

Advisory Committees ◽

Healthy Donors

Abstract Abstract 565 Background: The interaction of myeloma (MM) cells with bone marrow accessory cells induces genomic, epigenomic and functional changes which promote tumor development, progression, cell adhesion mediated-drug resistance (CAM-DR), and immune suppression. As in other cancers, bidirectional interaction between MM cells and surrounding cells regulates tumor development on the one hand, while transforming the BM microenvironment into a tumor promoting and immune suppressive milieu on the other. Recent developments in targeted therapies have indicated that generation of the most effective therapeutic strategies requires not only targeting tumor or stroma cells, but also methods to overcome blockade of anti-tumor immune responses. In addition to lymphoid immune suppressor cells such as regulatory T cells (Tregs), distinct populations of myeloid cells such as myeloid derived suppressor cells (MDSCs) can effectively block anti-tumor immune responses, thereby representing an important obstacle for immunotherapy. While MDSCs are rare or absent in healthy individuals, increased numbers of MDSCs have been identified in tumor sites and peripheral circulation. Recent studies have in particular focused on MDSCs in the context of tumor promoting, immune suppressing, stroma in solid tumors. However, their presence and role in the tumor promoting, immune suppressive microenvironment in MM remains unclear. Methods: Here we assessed the presence, frequency, and functional characteristics of MDSCs in patients with newly diagnosed or relapsed MM compared to MM patients with response and healthy donors. We first identified a distinct MDSC population (CD11b+CD14−HLA-DR-/lowCD33+CD15+) with tumor promoting and immune suppressive activity in both PB and BM of MM patients. Moreover, we determined the immunomodulatory effects of lenalidomide and bortezomib on induction of MDSCs by MM cells, as well as on MDSC function. Results: MDSCs were significantly increased in both PB and BM of patients with active MM compared to healthy donors and MM in response (p<0.01). To determine whether the CD11b+CD14−HLA-DR-/lowCD33+CD15+ myeloid cell population represents functional MDSCs, we first assessed tumor promoting role of MDSCs in the MM microenvironment by culturing MM cell lines with MM patient bone marrow stroma cells (BMSC), with or without depletion of MDSCs. Importantly, BMSC-mediated MM growth decreased to baseline levels of MM cells alone when MDSCs were removed from the BMSC microenvironment. Moreover, MDSCs isolated from MM-BM using magnetic-Ab and/or FACS sorting cell separation, directly induced MM cell growth and survival, evidenced by 3H-thymidine incorporation and MTT assays. Since the interaction between tumor and stromal accessory cells is bidirectional, we next analysed the impact of MM cells on MDSC development. Importantly, MM cell lines cultured with PBMCs from healthy donors induced a 7 fold increase in MDSCs. We also examined the immune suppressive functions of MDSCs in cultures of autologous T cells with T cell stimulators, in the presence and absence of MDSCs from MM-PB or MM-BM. Freshly isolated MDSCs from both MM-PB and MM-BM induced significant inhibition of autologous T cell proliferation. Moreover, MDSC-associated immune inhibitory molecules arginase-1 (ARG-1) and reactive oxygen species (ROS), as well as inhibitory cytokines IL-6 and IL-10, were significantly increased in BM MDSCs, evidenced by intracellular flow cytometry analysis. In addition, MM BM MDSCs induced development of Treg from autologous naïve CD4+T cells. Finally, we analysed whether MDSCs impacted response to bortezomib and lenalidomide. Culture of MDSCs with MM cell lines, with or without bortezomib (5nM) and lenalidomide (1uM), demonstrated that less MM cell cytotoxicity in the presence of MDSCs. Conclusions: Our data show that MDSCs are increased in the MM microenvironment and mediate tumor growth and drug resistance, as well as immune suppression. Therefore targeting MDSCs represents a promising novel immune-based therapeutic strategy to both inhibit tumor cell growth and restore host immune function in MM. Disclosures: Raje: Onyx: Consultancy; Celgene: Consultancy; Millennium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding. Munshi:Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy. Richardson:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees.

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Lymphocyte and Hematopoietic Stem and Progenitor Cell (HSPC) Subsets Mobilized By Either Plerixafor or G-GCSF: A Retrospective Comparison of Grafts Harvested in Healthy Allogeneic Stem Cell Donors

Blood ◽

10.1182/blood.v124.21.2451.2451 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2451-2451 ◽

Cited By ~ 1

Author(s):

Georgine E. De Greef ◽

Eric Braakman ◽

Wendimagegn G Alemayehu ◽

Larissa De Graaf ◽

Peter van Geel ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

The Netherlands ◽

Board Of Directors ◽

T Cell Subsets ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Healthy Donors ◽

Cd34 Cells

Abstract Plerixafor (PXF) is a bicyclam molecule, which acts as a reversible inhibitor of SDF-1 binding to CXCR4. A single injection results in immediate release of CD34+ cells into the peripheral blood. Sofar, PXF has been used for stem cell mobilization only in a limited number of allogeneic donors (Devine et al. Blood.2008;112(4):990) The currently ongoing randomized phase 2 Hovon -107 study of the Dutch hemato-oncology group HOVON (www.hovon.nl) aims to compare the feasibility of intravenous (iv) versus subcutaneous (sc) PXF (Genzyme Europe BV) 320 µg/kg subcutaneously (sc) 9 hours before the planned stem cell collection or intravenously (iv) 4 hours before stem cell collection in healthy adult matched sibling donors. Concurrently, all stem cell products are evaluated for the total number of CD45+; CD34+ cells and other hematopoietic stem cell subsets, including more primitive progenitor cells (MPP/CMP: CD34+/CD45RA-/CD90- and HSC :CD34+/CD45RA-/CD90+). Furthermore, the frequency and absolute numbers of CD3+, CD4+; CD8+;CD19+; CD 3-CD16/CD56+ (NK) cells and several T cell subsets, including Foxp3+, Th1, Th2 and Th17 cells, are assessed. Thereby, the HOVON-107 study enabled us to retrospectively compare lymphocyte and CD34+ HSPC subsets in grafts harvested in healthy donors (n=27) following PXF versus a similar evaluation of those subsets in grafts (n=10) harvested following G-CSF(Neupogen) (2 x 500 ug/kg (sc) for 5 days). Data are presented with respect to the composition of stemcell harvests, obtained after a single gift of PXF (13 iv and 14 sc) followed by 15 liters leucopheresis. For comparison of the stem cell products between the two groups the Mann-Whitney U test was applied. Results: Both groups are comparable with respect to age/sex. Mobilization with PFX resulted in similar WBC numbers as compared to G-CSF mobilization. The total number of CD34+ cells was significantly lower after PFX mobilization: median 200 x106; (range 40-560) vs 400 x106 (360-840) after G-CSF (p=0.000). However after PFX mobilization, the CD34+ cells contained a higher frequency of immature HSC and a lower frequency of MPP as compared to G-CSF mobilized grafts. The lower number of CD34+ HSPC and the higher frequency of HSC within CD34+ HSPC resulted in similar numbers of immature HSC in PXF mobilized grafts (PFX 50;1-218 x106 for G-CSF 90;11-200 x106 p=0.411).Although it is known that Plerixafor can mobilize a higher number of T-cells no data are available about the frequencies of distinct T cell subsets in the grafts. PFX mobilization resulted in higher numbers of CD3+T cells and CD19+B cells. The number of CD3-CD16/56+ NK-cells did not differ between both groups. Within the CD3+ T cell population, the CD4/CD8 ratio did not differ between both groups of mobilized grafts. While absolute numbers of T-cells were significantly increased, the frequencies of IFN-gamma+ Th1 cells, IL-4+ Th2 cells; IL-17+ Th17 cells and Foxp3+ regulatory T cells were not significantly different between both groups, resulting in increased Treg and Th1 after PFX (see Table below) In conclusion, allogeneic stem cell grafts harvested in healthy donors following a single dose of Plerixafor contain higher numbers of primitive progenitor cells, and higher numbers of both effector and regulatory T-cells as compared to grafts harvested following G-CSF. The impact of altered subset numbers on clinical endpoints including graft versus host, engraftment, and overall outcome remain to be established. Abstract 2451. TableCD3 (x 109)CD3/4 (x 109)CD3/8 (x 109)CD19 (x 109)CD3-CD16/56+ (x 109)Treg (x 109)Th1 (x 109)Th2 (x 109)Th17 (x 109)PFXMedian Range22.7 9.8-56,7 13.2 6.3-30.56.6 2.8-22.15.7 0.6-18.11.40.5-4.30.7 0.3-2.52.8 0.3-9.30.2 0.0-1.80.20.0-6.8G-CSFMedian Range12.8 7.6-217.5 4.3-15.43.8 2.0-6.03.1 1.9-4.51.30.5-2.90.4 0.2-1.20.9 0.4-2.70.20.1-0.40.1 0.0-0.5P-value0.0010.0050.0030.0020.7320.0220.0160.2890.129 Disclosures De Greef: Sanofi The Netherlands: Membership on an entity's Board of Directors or advisory committees. Petersen:Sanofi the Netherlands: Membership on an entity's Board of Directors or advisory committees. Visser:Sanofi the Netherlands: Membership on an entity's Board of Directors or advisory committees. Niederwieser:Novartis, Gentium, Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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